Beta-catenin is a key activator of Wnt signaling target genes. Aberrant activation of beta-catenin through mutational inactivation of its negative regulators has been implicated in the pathogenesis of several common human cancers. Recent studies from our laboratory have demonstrated that beta-catenin is subject to an autofeedback regulation after the adenomatous polyposis coli (APC) tumor suppressor protein mediated regulatory mechanism failed due to mutation. Functional screening of a cDNA library constructed by enriching beta-catenin induced genes through subtraction has lead to the identification the scaffolding protein Axin2, a target gene of beta-catenin. A homolog of Conductin or mouse Axil, Axin2 was previously identified as a negative regulator of Wnt/beta-catenin signaling by several other laboratories. Our transient expression experiments revealed a novel function of Axin2 in the destruction of beta-catenin that is uniquely different from that of the APC-mediated regulatory mechanism. We hypothesize that auto-feedback regulation of beta-catenin by Axin2 is an important mechanism of canonical Wnt/beta-catenin signaling. Based on this hypothesis, we suggest: 1) downregulation of beta-catenin requires a synergetic effect contributed by both APC and Axin 2; and 2) failure of Axin2-mediated auto-feedback regulation will lead to aberrant activation of beta-catenin and consequently neoplastic transformation. We propose to test this hypothesis using a combination of genetic and biochemical approaches as outlined in the following specific aims. AIM I: To assess whether synergistic cooperation between APC and Axin2 is required for the downregulation of beta-catenin using a set of genetically engineered colon cancer cell lines as the in vitro model system; and AIM II: To test whether loss of the auto-feedback function of Axin2 will cause aberrant activation of beta-catenin and tumorigenesis in vivo using genetically engineered mutant mice harboring heterozygous Axin2 allele. AIM III: To define the contribution of individual protein domains to the Axin2 tumor suppressor function, and use these information to delineate the unique features in Axin2-mediated destruction of beta-catenin. The long term goal of these proposed studies is to gain new mechanistic information concerning the pathogenesis of beta-catenin in human cancer, with the objective of identifying key cellular targets useful for rational cancer prevention and therapy.